Abstract About 257 million people worldwide are chronically infected with hepatitis B virus (HBV). Chronic HBV infection is the main risk factor of hepatocellular carcinoma (HCC). There is no cure for HBV. None of anti-HBV drugs (these include interferon and nucleos(t)ide analogs (NAs) that inhibit HBV reverse transcription) work for all infected individuals. All anti-HBV drugs failed to achieve much desired loss of serum surface antigen of HBV (HBsAg) in most cases. One of the most difficult problems in treating patients with chronic hepatitis B is the lack of clear predictors of when it is safe to stop antiviral therapy. In fact, clinical practice guidelines on management of chronic HBV infection consider identification of the markers to predict successful discontinuation of anti-HBV therapy with NAs as a major unmet need. For HBV e-antigen (HBeAg)-positive patients, a seroconversion is routinely used to attempt to stop therapy but for HBeAg-negative patients no such marker exists. A desire to avoid lifelong therapy has dissuaded some patients from treatment. We made several relevant findings. Using paired liver/HCC tissues from untreated chronic HBV carrier humans we found that regardless of ongoing HBV replication, the HBV integrant-derived RNAs (id-RNAs) that bear the 3'-end insert of the host sequence next to the poly(A) tail accumulated in all tissues and were relatively abundant in 60% of the tissues, while HBV replication-derived RNAs (rd-RNAs) were the least abundant species in 70% of tissues. The data suggest that id-RNAs coding for the envelope proteins (i.e., HBsAg) may produce significant fraction of HBsAg in cells bearing HBV DNA integrants. Therefore, anti-HBV drugs fail to achieve the loss of HBsAg, because major amounts of HBsAg could be produced from id-RNAs regardless of HBV replication. We thus hypothesize that a considerable fraction of patients that underwent prolong anti-HBV therapy and remain HBsAg-positive will likely have only id-RNAs accumulated in the livers, but not the rd-RNAs. However, no studies were conducted to evaluate how anti-HBV treatment alters the id-RNAs/rd-RNAs ratio. We will examine if intrahepatic id-RNAs and rd-RNAs can be used as biomarkers for evaluation of NA therapy (Aim 1). Using liver tissue samples from HBeAg(-)/HBsAg(+) patients that underwent NA therapy prior to liver transplantation, we expect to find that id-RNAs' abundance is common for chronic HBV infection, id-RNAs are a sizeable source of HBsAg that cannot be eliminated by NAs, and reduction of rd-RNAs' levels adequately reflects suppression of HBV replication by NAs. Next, considering (i) that blood draws (and not biopsies) are frequent for chronic HBV carriers, and (ii) recent reports on HBV RNA in sera, we will use sera from HBeAg(-)/HBsAg(+) patients that underwent prolong NA therapy and stopped the treatment, and find if serum rd-RNAs can be used to determine the time when to safely stop NA therapy (Aim 2). We hypothesize that serum rd-RNAs' levels adequately reflect intrahepatic HBV replication and efficiency of NA therapy, and NA therapy could be stopped (so there will be no permanent HBV rebound) when rd-RNAs' levels are undetectable/profoundly suppressed.